210 6.3  Optical Force Tools

used on single DNA molecules in vitro, for example, in the study of DNA replication. This

technique of extending a biomolecule-​tethered bead by flow can also be used in conjunction

with optical and magnetic tweezers to facilitate the initial stable trapping of the bead.

The molecular combing technique can be adapted to significantly improve throughput.

This is seen most dramatically in the DNA curtains technique (Finkelstein et al., 2010). Here,

DNA molecules are tethered on a nanofabricated microscope coverslip containing etched

platforms for tether attachment such that the tethered end of a molecule is clear from the

coverslip surface, thus minimizing the effects of surface forces on the molecule, which are

often difficult to quantify and can impair the biological function of DNA. Optimization of the

tethering incubation conditions allows several individual DNA molecules to be tethered in

line, spaced apart on the coverslip surface by only a few hundred nanometers.

The molecules can be visualized by labeling the DNA using a range of DNA-​binding dyes

and imaging the molecules in real time using fluorescence microscopy. This can be used to

investigate the topological, polymer physics properties of single DNA molecules, but can also

be used in investigating a variety of different molecular machines that operate by binding to

DNA by labeling a component of the machine with a different color fluorophore and then

utilizing dual-​color fluorescence imaging to monitor the DNA molecules and molecular

machines bound to them simultaneously. The key importance of this technique is that it

allows several tens of individual DNA molecules to be investigated simultaneously under the

same flow and imaging conditions, improving statistical sampling, and subsequent biological

interpretation of the data, enormously.

6.3  OPTICAL FORCE TOOLS

There are several biophysical techniques that utilize the linear momentum associated with a

single photon of light to generate forces that then can be used to probe and manipulate single

biomolecules and even whole cells. Optical tweezers utilize this approach, as do the related

optical stretcher technology. Optical tweezers are an exceptionally powerful tool for manipu­

lating single biomolecules and characterizing many aspects of their force-​dependent features,

and for this reason we explore the theory of their operation in detail here. Although single

biomolecules themselves cannot be optically trapped with any great efficiency (some early

optical tweezers experiments toyed with rather imprecise manipulation of chromosomes),

they can be manipulated via a micron-​sized optically trapped bead. But there are also

methods that can utilize the angular momentum of photons to probe the rotary motion of

the biological material. Other applications of optics, which allow monitoring of biological

forces, include Brillouin scattering, polarization microscopy, and Förster resonance energy

transfer (FRET).

6.3.1  BASIC PRINCIPLES OF OPTICAL TWEEZERS

The ability to trap particles using laser radiation pressure was reported first by Arthur Ashkin,

the pioneer of optical tweezers (also known as laser tweezers) (Ashkin, 1970). This was a rela­

tively unstable 1D trap consisting of two juxtaposed laser beams whose photon flux resulted

in equal and opposite forces on a micron-​sized glass bead. The modern form of the standard

3D optical trap (specifically described as a single-​beam gradient force trap), developed in the

1980s by Ashkin et al. (1986), results in a net optical force on a refractile, dielectric particle,

which has a higher refractive index than the surrounding medium, roughly toward the inten­

sity maximum of a focused laser. These optical force transduction devices have since been

put to very diverse applications for the study of single-​molecule biology (for older but still

rewarding reviews, see Svoboda and Block, 1994 for an accessible explanation of the physics,

and Moffitt et al., 2008 for a compilation of some of the applications).

Photons of light carry linear momentum p given by the de Broglie relation p =​ E/​c =​ hν/​

c =​ h/​λ, for a wave of energy E, frequency ν, and wavelength λ where c is the speed of light and

h Plank’s constant. Photon momentum results in radiation pressure if photons are scattered

KEY BIOLOGICAL

APPLICATIONS:

RHEOLOGYTOOLS

Molecular separation and

identification.